Identification of sunflower genotypes using microsatellite markers

Polymorphism of some DNA microsatellite sequences of sunflower breeding genotypes (inbred lines, hybrids) was investigated. Allelic composition on 8 SSR loci is unique characteristic of every analysed genotype allowing their identification. Microsatellite markers are proposed to be used for definition of line genetic purity and of F1 seeds hybridity range.     

Genetic diversity and geographical dispersal in grapevine clones revealed by microsatellite markers.

Intravarietal genetic diversification associated with geographical dispersal of a vegetatively propagated species was studied using grapevine Vitis vinifera L. 'Cabernet Sauvignon' as a model. Fifty-nine clonal samples obtained from 7 countries (France, Chile, Spain, Australia, Hungary, USA, and Italy) were analyzed using 84 microsatellite markers. Eighteen polymorphic microsatellite loci (21.4%) were detected, finding 22 different genotypes in the population analyzed with a genetic similarity of over 97%. The presence of chimeric clones was evidenced at locus VMC5g7 by means of a segregation analysis of descendants by self-pollination of a triallelic Chilean clone and by somatic embryogenesis analysis, showing a mutation in L2 cell layer. Only 2 clones (obtained from France and Australia) presented the ancestral genotype, and the most divergent genotype was exhibited by another French clone, which had accumulated 5 somatic mutations. The 2 largest populations considered (from France and Chile) showed a clear divergency in the polymorphisms detected. These antecedents enabled the tracing of geographical dispersal with a phylogenetic hypothesis supporting France as the center of origin of diversification of Cabernet Sauvignon. The results obtained could help to explain diversification processes in other grapevine cultivars. The possibility that this kind of genetic variability occurs in other vegetatively propagated species is discussed, focusing on possible fingerprinting applications.     

Development of microsatellite markers for the grapevine fungal pathogen Phaeomoniella chlamydospora

Twenty polymorphic microsatellite markers from microsatellite-enriched genomic DNA of the grapevine fungal pathogen, Phaeomoniella chlamydospora, were developed and characterized. The markers were used to genotype isolates from Australia and from Europe/Eurasia. The number of alleles per locus ranged from two to 11. Gene diversity per locus ranged from 0.08 to 0.63 among Australian isolates, and from 0.2 to 0.77 among isolates from Europe/Eurasia, demonstrating the suitability of these markers for population genetic studies of P. chlamydospora. Eighteen of the 20 markers also amplified a product in the closely related Phaeoacremonium aleophilum.     

Characterization of Spanish grapevine cultivar diversity using sequence-tagged microsatellite site markers.

A broad germplasm bank collection containing most of the autochthonous Spanish grapevine cultivars was analyzed using six sequence-tagged microsatellite site (STMS) loci: VVS2, VVMD5, VVMD7, ssrVrZAG47, ssrVrZAG62, and ssrVrZAG79. The number of alleles obtained ranged from 9 in ssrVrZAG47 to 13 in VVS2, and the observed genotypes per locus varied between 24 (ssrVrZAG47) and 41 (VVSS2). A total of 57 unique genotypes were obtained considering all 6 loci, and 40 varieties presented at least 1 of these specific genotypes. The genotypic combinations for the 6 loci have generated 163 different profiles in the 176 cultivars. Ten pairs of accessions and one group of four Garnacha's cultivars can not be differentiated. The observed heterozygosity varied between 75.6 (VVMD7) and 90.9% (VVMD5), without significant differences from the expected values for any loci. The VVMD5 locus was the most informative, and also showed the highest discrimination power. The cumulative discrimination power for all six loci was practically 1; however, in fact, these STMS loci have differentiated only about 93% of the accessions, probably owing to high relatedness of the plant material. Usefulness of this STMS set for characterization of a Spanish grapevine collection is emphasized, as well as the elaboration of databases with these molecular markers.     

Genetic neighbourhood of clone structures in eelgrass meadows quantified by spatial autocorrelation of microsatellite markers

Limited dispersal distances in plant populations frequently cause local genetic structure, which can be quantified by spatial autocorrelation. In clonal plants, three levels of spatial organization can contribute to positive autocorrelation; namely, the neighbourhood of (a) ramets, (b) clone fragments and (c) entire clones. Here we use data from an exhaustive sampling scheme on a clonal plant to measure the contribution of the neighbourhoods of each distinct clonal structure to total spatial autocorrelation. Four plots (256 grid points each) within dense meadows of the marine clonal plant Zostera marina (eelgrass) were sampled for clone structure with nine microsatellite markers ( approximately 80 alleles). We found significant coancestry (f(ij)), at all three levels of spatial organization, even when not allowing for joins between samples of identical genets. In addition, absolute values of f(ij) and the maximum distance with significant positive f(ij) decreased with the progressive exclusion of joins between alike genotypes. The neighbourhood of this clonal plant thus consists of three levels of organization, which are reflected in different kinship structures. Each of these kinship structures may affect the level of biparental inbreeding and the physical distance between flowering shoots and their outcrossing neighbourhood. These results also emphasize the notion that spatial autocorrelation crucially depends on the scale and intensity of sampling.     

Reconstruction of a grapevine pedigree by microsatellite analysis

Microsatellites are ideal markers for revealing genetic relationships between individuals because of their co-dominant inheritance. In this study we determined the genetic profiles of 52 grapevine cultivars using 32 microsatellite markers. We were able to define the complex genetic relationship among nine European grapevine cultivars. None of these parent-offspring combinations were anticipated beforehand. The ancient cultivar Silvaner is shown to be an offspring from Traminer and Österreichisch Weiß. Rotgipfler originates from a cross between Traminer and Roter Veltliner, while Frühroter Veltliner originates from Roter Veltliner×Silvaner and Frühroter Veltliner× Portugieser gave rise to Jubiläumsrebe. A pedigree illustrating the putative crosses was reconstructed.     

Genetic relationships among wheat genotypes, as revealed by microsatellite markers and pedigree analysis

Genetic relationships among 20 elite wheat genotypes were studied using microsatellite markers and pedigree analysis. A total of 93 polymorphic bands were obtained with 25 microsatellite primer pairs. Coefficient of parentage (COP) values were calculated using parentage information at the expansion level of 5. The pedigree-based similarity (mean 0.115, range 0.00-0.53) was lower than the similarity assessed using microsatellite markers (mean 0.70, range 0.47-0.91). Similarity estimates were used to construct dendrograms by using the unweighted pair-group method with arithmetic averages (UPGMA). Clustering of genotypes in respect of marker-based similarity revealed two groups. Genotype PBW442 diverged and appeared as distinct from all other genotypes in both marker-based and pedigree-based analysis. The correlation of COP values with genetic similarity values based on microsatellite markers is low (r = 0.285, p < 0.05). The results indicate a need to develop wheat varieties with a diverse genetic background and to incorporate new variability into the existing wheat gene pool.     

Assessment of genetic variation and differentiation of hop genotypes by microsatellite and AFLP markers.

Microsatellites have many desirable marker properties and have been increasingly used in crop plants in genetic diversity studies. Here we report on the characterisation of microsatellite markers and on their use for the determination of genetic identities and the assessment of genetic variability among accessions from a germplasm collection of hop. Thirty-two polymorphic alleles were found in the 55 diploid genotypes, with an average number of eight alleles (3.4 effective alleles) for four microsatellite loci. Calculated polymorphic information content values classified three loci as informative markers and two loci as suitable for mapping. The average observed heterozygosity was 0.7 and the common probability of identical genotypes was 3.271 x 10(-4). An additional locus, amplified by one primer pair, was confirmed by segregation analysis of two crosses. The locus discovered was heterozygous, with a null allele in the segregating population. The same range of alleles was detected in nine triploid and five tetraploid hop genotypes. Cultivar heterozygosity varied among all 69 accessions, with only one cultivar being homozygous at four loci. Microsatellite allele polymorphisms distinguished 81% of all genotypes; the same allelic profile was found mainly in clonally selected cultivars. Cultivar-specific alleles were found in some genotypes, as well as a specific distribution of alleles in geographically distinct hop germplasms. The genetic relationship among 41 hop accessions was compared on the basis of microsatellite and AFLP polymorphisms. Genetic similarity dendrograms showed low correlation between the two marker systems. The microsatellite dendrogram grouped genetically related accessions reasonably well, while the AFLP dendrogram showed good clustering of closely related accessions and, additionally, separated two geographically distinct hop germplasms. The results of microsatellite and AFLP analysis are discussed from the point of view of the applicability of the two marker systems for different aspects of germplasm evaluation.     

Identification of new microsatellite markers in Panax ginseng

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The (AG)n motif was most common (23.1%), followed by the (AAC)n motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.     

Identification of microsatellite markers in the rye genome

The rye genomic library, which consists of DNA fragments in the range of 0.5–1.1 kb, was screened for the presence of tri-and tetranucleotide and compound microsatellites. Of the 1,600,000 clones analysed, 102 clones were positive and 41 were suitable for SSR primer pair design. Twenty-six primer pairs amplified specific products, and six of them were capable of detecting polymorphism among 30 rye accessions of different genetic backgrounds. Using a set of Chinese Spring-Imperial wheat-rye addition lines, it was possible to locate 3 newly identified microsatellites on chromosomes 3R, 4R and 7R.     

Identification and registration of corn genotypes using molecular markers

In plant genetics and breeding, second-generation molecular markers allow detailed characterization of plant genotypes. Unique genotypes at ten simple sequence repeat (SSR) loci were established for 40 maize accessions by means of PCR. For every locus, SSR analysis revealed heterozygotes among simple hybrids, which made it possible to identify the parental forms with a high probability of exclusion of nonparental forms. A system was proposed for registration of maize genotypes in the form of genetic formulas reflecting the allelic state of microsatellite loci, in order to catalog, preserve, and effectively employ the existing maize gene pool in breeding.     

Genetic homogeneity of in vitro raised plants of grapevine cv. Crimson Seedless revealed by ISSR and microsatellite markers

The present study was conducted to test the clonal homogeneity of six month old tissue culture raised plants of grapevine cv. Crimson Seedless using Inter Simple Sequence Repeat (ISSR) and Simple Sequence Repeat (SSR) markers. Visible assessment of these in vitro raised plants maintained in polyhouse did not show any morphological differences among themselves. However, to test the genetic homogeneity of these plants, we screened 50 ISSR primers out of which, 22 primers produced scorable and repeatable bands. These 22 primers were used further for assessing genetic homogeneity of in vitro raised plants of Crimson Seedless. These 22 ISSR primers generated 134 distinct band classes with a total of 3216 scorable bands. All the primers showed uniform banding pattern for all the in vitro raised plants and the mother plant. In case of 5 SSR primers (VS1, VVMD5, VVS2, VMCNG4c8 and VVMD31) used, a total of 288 scorable bands were obtained. The allele sizes ranged from 98 to 254 bp. Allelic composition of 23 in vitro raised plants and the mother plant at 5 SSR loci did not show any polymorphism. The results of the two marker systems in the present study revealed the genetic uniformity among the in vitro raised plants demonstrating the reliability of in vitro propagation system used for the cultivar.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.     

Identification of salmonid fish using microsatellite markers with identical PCR-primers

A set of homologous primers were designed to provide the amplification of microsatellite markers in a variety of salmonid species. They can help to solve problems associated with the genetics and conservation biology of salmonids, viz., (1) species identification; (2) determination of interspecific hybrids; (3) genetic estimation of salmonid biodiversity in a watershed; (4) individualization of tissue samples, i.e., testing on their belonging to the same individual; and (5) identification of a fish’s origins.     

The use of microsatellite DNA markers for soybean genotype identification

Conventional morphological and pigementation traits, as well as disease resistance, have been used to distinguish the uniqueness of new soybean cultivars for purposes of plant variety protection. With increasing numbers of cultivars and a finite number of conventional characters, it has become apparent that such traits will not suffice to establish uniqueness. The objective of this work was to provide an initial evaluation of microsatellite or simple-sequence-repeat (SSR) DNA markers to develop unique DNA profiles of soybean genotypes. Microsatellites are DNA sequences such as (AT) _n /(TA) _n and (ATT) _n /(TAA) _n that are composed of tandemly repeated 2–5-basepair DNA core sequences. The DNA sequences flanking microsatellites are generally conserved allowing the selection of polymerase chain reaction (PCR) primers that will amplify the intervening SSR. Variation in the number of tandem repeats, n, results in PCR product length differences. The SSR alleles present at three (AT) _n /(TA) _n and four (ATT) _n /(TAA) _n loci were determined in each of 96 diverse soybean genotypes. Between 11 and 26 alleles were found at each of the seven loci. Only two genotypes had identical SSR allelic profiles and these had very similar pedigrees. The gene diversity for the seven markers averaged 0.87 for all 96 genotypes and 0.74 for a subset of 26 North American cultivars. These are much higher than soybean gene diversity values obtained using RFLP markers, and are similar to the average values obtained for human microsatellite markers. SSR markers provide an excellent complement to the conventional markers that are currently used to characterize soybean genotypes.